Corticothalamic Projection Neuron Development beyond Subtype Specification: Fog2 and Intersectional Controls Regulate Intraclass Neuronal Diversity

Abstract

Corticothalamic projection neurons (CThPN) are a diverse set of neurons, critical for function of the neocortex. CThPN development and diversity need to be precisely regulated, but little is known about molecular controls over their differentiation and functional specialization, critically limiting understanding of cortical development and complexity. We report the identification of a set of genes that both define CThPN and likely control their differentiation, diversity, and function. We selected the CThPN-specific transcriptional coregulator Fog2 for functional analysis. We identify that Fog2 controls CThPN molecular differentiation, axonal targeting, and diversity, in part by regulating the expression level of Ctip2 by CThPN, via combinatorial interactions with other molecular controls. Loss of Fog2 specifically disrupts differentiation of subsets of CThPN specialized in motor function, indicating that Fog2 coordinates subtype and functional-area differentiation. These results confirm that we identified key controls over CThPN development and identify Fog2 as a critical control over CThPN diversity.

Figure 1. CThPN-specific genes of high biological relevance for corticothalamic development classified based on temporal expression

A set of biologically particularly relevant genes is shown, selected from the larger group of differentially expressed genes (available at GSE61711) based on function in other systems and temporal match to relevant biological processes by CThPN. Each group is represented by a prototypical expression profile shown on the left. (A) General Identity Genes: enriched in CThPN at all stages of development; (B) Early Development Genes: highly expressed by CThPN early in development; (C and D) Intermediate Development and Late Development Genes: exhibit increasing levels of expression as CThPN develop; and (E) Genes excluded from CThPN: expressed at high levels in other cortical projection neuron subtypes, serving as negative markers for CThPN. Expression by ISH and graphic expression profiles are shown for all genes listed in bold, either in Figure 2 or Figure S1. Data are represented as mean ± SEM.

Figure 2. Expression analysis of CThPN-specific genes by in situ hybridization parallels the temporal expression results from microarrays

Expression analysis, including graphic expression profiles from microarray and ISH, of genes representing each category listed in Figure 1 (bold). Cxxc5 and Tle4 (A–F) are representative examples of “general identity genes”. Rad21 (G–J) and Fog2 (K–M) exemplify “early development” and “intermediate development” gene categories. Ror2 and Shb represent “late development genes” (N–S). (B, E, H, L, O, R) White arrowheads indicate expression in the cortical plate (CP), and black arrowheads indicate expression in the ventricular zone (VZ). Scale bars, 200 μm.

Figure 3. Fog2 is expressed by postmitotic corticothalamic projection neurons, but is excluded from corticospinal and callosal projection neurons

(A–C‴) FOG2 ICC (red) on coronal sections from brains injected with FluoroGold (FG) in thalamus, spinal cord, or contralateral cortex to retrogradely label CThPN, CSMN, or CPN, respectively (green). Schematic views of FG injection sites are at the top of each panel group. (A-A‴) FOG2 co-localizes with FG in retrograde labeled CThPN (white arrowheads in A′-A‴, area boxed in A). FG-labeled CSMN (B-B‴), or CPN (C-C‴) do not express FOG2. (D–G) Time-course of FOG2 expression revealed by ICC. FOG2 is first expressed at E12.5 in the preplate (white arrowheads) (D). FOG2 is strongly expressed in deep cortical plate during embryonic development (E), and in layer VI during the first postnatal week (white arrowheads) (F, G). Expression in the cingulate cortex is detected postnatally (open arrowheads F–G) Scale bars, 50 μm (A, B, C), 20 μm (A′-A‴, B′-B‴, C′-C‴), 50 μm (D–E), 200 μm (F), 500 μm (G).

Figure 4. Loss of Fog2 function disrupts axonal targeting of CThPN in the frontal cortex

Coronal sections of brains injected with FG in discrete thalamic nuclei: VA/VL (A–B″), MD (E–F″), and VB (I–J′). Boxed areas in (A, B, E, F, I, J) are magnified in (A′–J′). Schematic of the nuclei injected, and the cortical areas where retrograde labeled CThPN were analyzed (C, G, K). Quantification of labeled CThPN in the dorsal and lateral aspects of motor cortex from each nucleus (D, H, L). Asterisk indicates statistical significance (p<0.05, t-test). Scale bar, 500 μm (A–J), 50 μm (A′–J′). Data are represented as mean ± SEM.

Figure 5. In the absence of Fog2 function, CThPN are born and migrate normally, but aberrantly up-regulate Ctip2 expression in motor cortex

(A–F′) CTIP2 and BrdU ICC on P6 motor cortex. More layer VI neurons BrdU-pulsed at E12.5 express high level Ctip2 in motor cortex of Fog2 cKO mice. (G) Quantification of BrdU pulsed neurons at E12.5 across the thickness of motor cortex shows no differences in the number and distribution of CThPN between WT and Fog2 cKO. (H) Quantification of double labeled BrdU⁺-CTIP2⁺ neurons located in layers VI and V expressed as percentage of total BrdU⁺ pulsed neurons at E12.5. (I) Quantification of Ctip2 expression level in frontal cortex by qPCR analysis. Asterisk (p<0.05), two asterisks (p<0.01). (J–O′) CTIP2 and TBR1 ICC on P6 motor cortex. More neurons in Fog2 cKO motor cortex co- express high level Ctip2 and Tbr1. Scale bars, 100 μm (A–F), 50 μm (J–O), 20 μm (A′–F′, J′–O′). Data are represented as mean ± SEM.

Figure 6. Fog2 −/− neurons upregulate CTIP2 and project aberrantly to the ventral telencephalon

Electroporation of control^Gfp or Cre-recombinase^Gfp plasmids into Fog2^fx/fx embryos at E11.5. (A, E) Schematic of experimental approach, and diagram of the cortical and diencephalic regions shown with immunofluorescence. ICC for GFP, FOG2 and CTIP2 in control^Gfp (B′-B‴), and Cre^Gfp electroporated (F-F‴) neurons. In motor cortex, control GFP⁺ neurons express FOG2, and low level or no CTIP2 (open arrowheads, B-B‴). In contrast, many Cre^Gfp neurons express high level of CTIP2, and do not express FOG2 (white arrowheads, F-F‴). GFP ICC reveals axonal projections from electroporated neurons in the internal capsule en route to thalamus in control mice (C–D), and in the internal capsule, but aberrantly extending into the ventral hypothalamus, in experimental mice (G–H). Scale bars, 20 μm (B-B‴ and F-F‴) and 500 μm (C, G).

Figure 7. Fog2 mis-expression in SCPN reduces expression of Ctip2 and subcerebral axon projections, but does not induce projection to thalamus

(A) Schematic of experimental approach, and diagrams of the brain regions where electroporated neuron somata and axons were quantified. Electroporations always targeted motor cortex, but also spread into somatosensory cortex. (B) Quantification of corticothalamic axonal projections upon Fog2 mis-expression. The number of CTh axons is not significantly higher than the number layer VI-CThPN after electroporation at E13.5 (open arrowheads in C), in control or experimental conditions. (C–K) Fog2 mis- expression in layer V (between dashed lines) reduces expression of Ctip2 by SCPN. (G– K) The number of FOG2-GFP⁺ neurons expressing Ctip2 (white arrowheads) is significantly reduced compared to the number of tdTomato-only neurons expressing Ctip2 (open arrowheads). (K) For quantification, values are normalized to the ratio CTIP2/reporter in control tdTomato-only neurons (control CTIP2/tdTomato-only 100%, experimental CTIP2/GFP⁺ 46%). Asterisks indicate statistical significance (p<0.05). (L–O) Axons of SCPN mis-expressing Fog2 progress significantly less far in the cerebral peduncle than axons of control SCPN. Quantification of subcerebral projecting axons was performed at three levels: internal capsule (L), rostral cerebral peduncle (M), and caudal cerebral peduncle (N). Numbers of axons at each level are normalized to the initial number of axons in the internal capsule (O). Asterisk (p<0.05), two asterisks (p<0.01). Scale bars, 50 μm (C–F) and 20 μm (G–N). Data are represented as mean ± SEM.

Figure 8. FOG2 interacts with COUPTF1, GATA2, and GATA4 to regulate expression of Ctip2

(A–C) Co-expression of FOG2 and COUPTF1, GATA2, and GATA4 in the developing motor cortex identify by ICC for COUPTF1, GATA2, and GATA4 combined with ISH for FOG2 at E15.5. Examples of double positive cells are shown (arrowheads). (D–F) Co-IP of FOG2 with COUPTF1 (D), GATA2 (E), and GATA4 (F). Immunoblotts with Anti-Flag antibody detect Flag-COUPTF1, Flag-GATA2, and Flag-GATA4 proteins pulled-down by anti-HA antibody, but not by non-specific IgG. Arrow marks 50KDa on the ladder to estimate protein size. (G) Putative regulatory region upstream of Ctip2 used for luciferase reporter assays contains COUPTF1, GATA2, and GATA4 consensus binding sites (red, green, blue, respectively) and is highly conserved across mammals. (H) Percentage of luciferase activity relative to the baseline (pGL3-Ctip2-Luciferase alone) in the presence of FOG2, COUTF1, COUPTF1 + FOG2, GATA2, GATA2 + FOG2, GATA4, GATA4 + FOG2. Asterisks indicate significance (t-test; p<0.05). Data are represented as mean ± SEM. (I) Schematic depicting CThPN subtype development, throughout distinct differentiation stages, from subtype specification to functional specialization. CPN, SCPN, and CThPN are schematized within an n-dimensional ‘subtype identity space’ in which individual subtype identities are defined by a range of variables (e.g. neuron morphology, axonal projections, circuit connectivity, three-dimensional spatial location, gene expression, the time of function of molecular controls, and other characteristics). This n-dimensional subtype identity is controlled by distinct transcription factors and other controls acting at progressive differentiation stages (e.g. early subtype specification, orange ovals; later subtype differentiation, purple ovals; some act both early and late, dual color ovals). Controls regulating identity development form key nodes of a transcriptional network, which is beginning to be elucidated. Fog2 is depicted in relationship to other key controls, regulating CThPN differentiation and diversity. Controls acting at the emergence of subtype identity can function in multiple, progressively more complex dimensions (e.g. wiggly lines depict extension into a representation of the CThPN spatial dimension, in which Fog2 functions differentially, repressing Ctip2 expression in dorso-medial CThPN subsets in combination with intersecting factors that might not be specific only to the CThPN subtype context). Basic elements of the left and middle sections of Panel I are modified with permission from Greig et al., 2013.